Composition, formulae, devices and methods for control of specificity and inclusivity of microorganisms containing closely related antigen epitopes

ABSTRACT

Compositions, formulae, devices and methods for the detection of target microorganisms, such as by visual immunoprecipitate assay, enzyme linked immunoassay, chemiluminescence, immunoblotting, or similar detection technology, wherein detection requires the discrimination among closely related genera, species and strains of antigenically related microorganisms based on immunological reactivity of a highly conserved antigen epitopes with a reagent system comprised of an antibody linked to a detecting reagent. The invention permits a detectable event to occur by exposing inaccessible but highly conserved and specific antigen epitopes to the detecting reagent. Exposure of such antigen epitopes without inactivating microbial metabolism allows for specific detection.

CROSS-REFERENCE TO RELATED APPLICATION

This application is claims priority from U.S. Provisional ApplicationSer. No. 60,096,566, filed Aug. 14, 1998.

TECHNICAL FIELD

The present invention relates generally to compositions, formulae,devices, and methods for detecting and identifying microorganisms and,more particularly, to compositions, formulae, devices, and methods fordetecting microorganisms by exposing highly conserved antigenic sites oftarget microorganisms without inactivating the microorganisms' abilityto grow to sufficient levels to be detected by an antibody linkeddetection system.

BACKGROUND OF THE INVENTION

Microbial diseases have long been a major health concern worldwide.Significant increase in the frequency and severity of outbreaks haveoccurred throughout the world. New pathogenic bacteria, such as E. coli0157:H7, have been identified. Furthermore, previously recognizedpathogenic genera have mutated to form drug resistant highly infectiousstrains such as Salmonella typhimirium DT 104. A key feature in theprevention of such diseases is early diagnosis. Epidemiologists mustlook for microbial contamination in the environment as well as in foodproducts to find the effective disease prevention strategies.

One example is the outbreak in 1992 of Enterohemorrhagic E. coli (EHEC)in the Pacific Northwest of the United States due to contaminated groundbeef. EHEC is a relatively “newly discovered” pathogen. EHEC was firstisolated in 1975, and it was not until 1982 that E. coli 0157:H7 wasassociated with two food related outbreaks of hemorrhagic colitis in theUnited States. The reported incidence of E. coli 0157 :H7 cases isincreasing. Typically, E. coli strains are harmless commensals, but afew strains are pathogenic. EHEC is particularly virulent and cantrigger deadly complications, including severe abdominal cramps andacute renal failure in children as well as cardiovascular and centralnervous system problems.

As another example, Salmonella is the leading cause (more than 50%) oftotal bacterial foodborne disease outbreaks, according to the UnitedStates Centers for Disease Control (CDC) surveillance of foodbornediseases. More than 40,000 cases per year were reported to the CDCduring the period 1988-1992. Salmonella can infect a broad variety ofwarm- and cold blooded animals, and can survive for long periods of timeoutside a host.

In a further example, Salmonella typhimurium DT 104 was first identifiedin the United Kingdom in the early 1990s. It is a highly adapted drugresistant strain of Salmonella known for its virulence. Resultingly,significant clinical interest has surrounded this serotype. S.typhimurium DT 104 contains core cell wall antigen epitopes that arehighly conserved among the genus Salmonella.

Listeria, a genus of gram positive bacteria, is widely distributed innature, having been isolated from soil, water, vegetation and manyanimal species. The detection frequency for Listeria in the agriculturalenvironment appears to be increasing. For specific outbreaks oflisteriosis, estimates place mortality at 30% to 40% of affectedpatients, however, little is known of the minimum infective dose. Oneparticularly troublesome aspect of Listeria control in foods is thatListeria can grow at temperatures as low as −0.4° C. and as high as 44°C. These factors all contribute to the increasing significance ofListeria as a food pathogen.

Campylobacter jejuni and coli have recently been identified as the leadcauses of enteritis, especially from poultry sources. This has led to anincreased need to discriminate these two species from several otherCampylobacter species which are not human pathogens. This requires thedifferential selection of more specific cell wall membrane antigenepitopes.

The ability to monitor potential environmental and food sources ofmicrobial contamination quickly and easily, but with very highspecificity, would reduce the risk of human infection. Therefore, ananalytical method which affords high specificity, preferably combinedwith a device able to assay for microorganisms, including bacteria,yeasts, molds, fungi, parasites and viruses, that requires no special ortechnical equipment, can be performed in the field and does not requirespecial skills would be useful. In the case of foodborne bacterialcontamination, four of the major disease-related organisms areSalmonella, Listeria, EHEC and Campylobacter.

While there are a number of Salmonella, Listeria, and EHEC detectionmethods presently available, trained laboratory technicians and aminimum of 2-5 days are required to obtain test results by the standardcultural methods of analysis. New, more rapid methods are based on suchtechniques as enzyme linked immunoassay EIA), DNA hybridization,immunodiffusion, or growth/metabolism measurements. While taking muchless time than the cultural methods, these rapid tests still requireskilled technical training, a functional laboratory, and specializedequipment. These tests generally take a total of two or more days,including considerable hands-on time. Campylobacter detectionmethodology to date is technically intensive requiring fastidious mediaand environmental conditions, in addition to well-trained analysts.

Another recent technology in the diagnostic field involves lateral flowimmunoassays. Such tests have been developed for the detection of humanchorionic gonadotropin (hCG), and applied to pregnancy testing.Typically, a monoclonal or polyclonal antibody is immobilized in adiscrete band near the distal end of a solid carrier strip, called thedetection zone. Another amount of antibody is labeled with a detectionreagent such as an inorganic sol or dyed polystyrene particle. Thislabeled antibody is reversibly fixed near the proximal end of thecarrier strip. Upon hydration of the proximal end with a sample fluidpotentially containing the antigen, the antigen reacts with the labeledantibody and the complex passes through the zone of immobilizedantibody, forming a sandwich upon reacting with the immobilizedantibody. The capture of the chromogenic reagent-antigen complex causesthe formation of a visible signal in the detection zone.

Two major challenges must be addressed to distinguish pathogenicbacteria, as opposed to distinguishing hormones or other solublemolecular targets. These challenges are the need to detect all of thestrains of a pathogenic microorganism in the presence of numerousantigenically related organisms, with a low tolerance for false positiveresults and a very low, preferably zero, tolerance for false negatives.The second challenge is the physical size and heterogeneity of themicroorganism itself. A typical clinical diagnostic test, such as a testfor hCG in urine, is focused on detecting a single, small, unique entity(ie., a hormone) in a well characterized matrix (e.g., urine).Furthermore, the structure of the analyte (hCG) is defined and uniformin size and composition.

Pathogen detection, for example, a test for Salmonella, must distinguisha particular pathogenic strain from nonpathogenic strains of similarmicroorganisms, such as Citrobacter spp. and Enterobacter spp. Incontrast to the well-defined small size and structure of most hormonesor marker proteins, microorganisms are very large, their surfaces areheterogeneous containing many distinct antigen epitopes that can undergochanges, such as the phase-switching of Salmonella flagella.

In addition, the cell wall membrane of many microorganisms containantigen epitopes, such as lipopolysaccharides, which are repeated with ahigh degree of consistency within a given genus. These antigen epitopesserve as highly desirable targets for reaction with complimentaryspecific antibodies which, in turn, provides a method of high accuracywith low false positives. The ability to isolate and bind to antibodiesreacting with these highly conserved antigen epitopes is difficult,however, because they can be sterically hindered by O-antigenpolysaccharide chains. They are generally inaccessible because of aphenomenon known as steric interference. This steric interference isprovided by the surface antigen epitopes of the microorganism. Examplesof surface structures known to contribute to this interference aresurface proteins, group specific lypopolysaccharides, flagella, andcellular encapsulation.

Although, aggressive treatments are available which will expose interiorantigen epitopes, these treatments destroy cell viability and in manycases disrupt cellular integrity completely. Examples of such treatmentsare heat treatment (boiling or autoclaving) and chemical extraction(nitrous acid digestion). The significant shortcoming of theseextractions is that they result in death of the microorganism.Therefore, if the cell population had not reached a sufficientlydetectable level prior to inactivation, a negative determination willresult.

Thus, there is a need in the art for methodologies that will allow thesimultaneous exposure of highly conserved masked antigen epitopes whilestill allowing the microorganisms to multiply. Further, there is a needin the art to incorporate improved selectivity for highly conservedtarget antigen epitopes of specific species in a population ofheterogeneous microorganisms in a variety of matrices. The presentinvention provides these and other, related advantages.

SUMMARY OF THE INVENTION

The present invention generally provides a novel, antigenic epitopeexposing microorganism growth composition. In one aspect, the inventionprovides a composition comprising a general enrichment media and atleast one structure modifying organic chemical. In one embodiment, thestructure modifying organic chemical is 2,4-dinitrophenol or carbonylcyanide-m-chlorophenyl hydrazone. In another embodiment, the structuremodifying organic chemical is 2,4-dinitrophenol. In yet anotherembodiment, the general enrichment media is selected from a variety ofreadily made or commercially available media including Terrific Broth,SOB medium, SOC medium, LB medium, NZCYM medium, minimal medium, lactosebroth, buffered peptone water, Brain Heart Infusion medium, Haemophilusbroth, tryptic soy broth, and nutrient broth.

It is another aspect of the present invention to provide a method fordetecting a microorganism in a test sample by contacting the test samplewith a composition comprising general enrichment media and at least onestructure modifying organic chemical, thereby forming a mixture. Thismixture is then incubated for a time sufficient to allow for detectablelevels of microorganisms to develop, after which the presence ofspecific microorganisms is detected. In one embodiment of this aspect ofthe invention the mixture is contacted with a detergent prior to orcontemporaneous with detection. In another embodiment, the mixture iscontacted with a detergent and heated prior to or contemporaneous withdetection. In one embodiment, the detergent is anionic. In yet anotherembodiment, the detergent is non-ionic. In certain embodiments, theanionic detergent may be selected from sodium dodecyl sulfate and sodiumdeoxycholate. In certain embodiments, the non-ionic detergent is NP-40,tergitol, or triton X-100. In certain other embodiments, the mixture isheated in the presence of the detergent to a temperature, between 40° C.and 121° C.

In certain embodiments of the detection method, the microorganismdetected is Listeria, Enterohemorrhagic E. coli, Salmonella, orCampylobacter.

Turning to another aspect of the invention, a method is provided fordetecting the presence of Listeria, Enterohemorrhagic E. coli,Salmonella, or Campylobacter in a test sample wherein the test sample iscontacted with a composition comprising general enrichment media and atleast one structure modifying organic chemical, followed by incubationof this mixture for a time sufficient to allow for detectable levels ofmicroorganisms to develop. Subsequent to the development of detectablelevels of microorganisms in the mixture, the presence of Listeria,Enterohemorrhagic E. coli, Salmonella, or Campylobacter is specificallydetected. In one embodiment of this aspect of the invention the mixtureis contacted with a detergent prior to or contemporaneous withdetection. In another embodiment, the mixture is contacted with adetergent and heated prior to or contemporaneous with detection. In oneembodiment, the detergent is anionic. In yet another embodiment, thedetergent is non-ionic. In certain embodiments, the anionic detergentmay be sodium dodecyl sulfate or sodium deoxycholate. In certainembodiments, the non-ionic detergent is NP-40, tergitol, or tritonX-100. In certain other embodiments, the mixture is heated in thepresence of the detergent to a temperature, between 40° C. and 121° C.

In another embodiment, the detection methodologies described hereinutilize an immunoassay. In certain embodiments, the immunoassay isselected from a visual immunoprecipitate assay, an enzyme linkedimmunoassay, chemiluminescence, and immunoblotting. In certain otherembodiments, the immunoassay is a visual immunoprecipitate assay. Alsoprovided in certain embodiments are immunoassays which utilize acomplementary monoclonal antibody, polyclonal antibody, or an antibodyfragment, wherein said antibody or antibody fragment is specific for ahighly conserved cell wall epitope in the target microorganism.

In another aspect of the invention, a method is provided, comprisingcontacting a test sample containing a microorganism with animmunoaffinity based detection device, wherein the test sample has beenpreviously propagated in the presence of a structure modifying organicchemical.

The invention also provides a method for propagating a microorganismsuch that cell wall antigen epitopes of the microorganism are altered bycontacting a test sample with a composition comprising generalenrichment media and at least one structure modifying organic chemical,and propagating the microorganism therein.

Turning to yet another aspect of the invention, a method for detectingmicroorganism specific epitopes on a target microorganism in a testsample is provided, comprising propagating a microorganism in a testsample in a permissive general enrichment media, wherein said mediacomprises a structure modifying organic chemical, and contacting thetest sample with a microorganism specific antibody linked to a detectingreagent, wherein reaction with the antibody indicates the presence ofthe microorganism. In further embodiment, contact between the testsample and the antibody occurs in device or assay system. In yet anotherembodiment, the assay system is selected from a visual immunoprecipitateassay, an enzyme linked immunoassay, chemiluminescence, andimmunoblotting. In another embodiment, the assay device is a lateralflow detection device. In certain embodiments, the antibody used in theabove methods is specific for a microorganism selected from Salmonella,Enterohemorrhagic E. coli, Listeria, and Campylobacter.

It is another aspect of the present invention to provide a lateral flowdevice for detecting a target microorganism in a sample comprising amicroorganism specific antibody and a test sample previously propagatedin a general enrichment media, the media comprising at least onestructure modifying organic chemical. In another embodiment of thisaspect of the invention the antibody is specific for any one ofSalmonella, Enterohemorrhagic E. coli, Listeria, or Campylobacter.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and examples. Inaddition, the various references set forth below describe in more detailcertain procedures or compositions (e.g., antibodies, detectionmethodologies, etc.), and are therefore each incorporated herein, byreference, in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

The term “antibody” as used herein includes polyclonal, monoclonal,humanized, chimeric, and anti-idiotypic antibodies, as well as fragmentsthereof such as F(ab′)₂ and Fab fragments and other recombinantlyproduced binding partners. Further, the antibodies may be covalentlylinked to or recombinantly fused to an enzyme, such as alkalinephosphatase, horse radish peroxidase, α-galactosidase, and the like.

“Structure modifying organic chemical” refers to an organic chemicalcapable of altering the composition of the cell wall of a microorganism,such that specific and conserved ligands are exposed. Briefly, suchorganic chemicals typically inhibit the transfer of stericallyinterfering epitopes to the cell wall. Such organic chemicals include,but are not limited to, 2,4-dinitrophenol, carbonylcyanide-m-chlorophenyl hydrazone or similar electron uncouplers (i.e.,disabling proton motive force), which have the effect of exposing highaffinity and specific epitopes which are recognized by monoclonal orpolyclonal antibodies.

The term “general enrichment media” refers to any media which is knownto be useful for facilitating the growth of microorganisms. Briefly, avariety of general enrichment media are commercially available and/orcan be readily made, these include, but are not limited to, Tryptonebased medium (e.g., Terrific Broth, SOB, SOC, and LB medium), NZCYMmedium, minimal medium, lactose broth, buffered peptone water, BrainHeart Infusion medium, Haemophilus broth, Tryptic Soy broth, Nutrientbroth and the like (see Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Press, 1989; Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing, 1995;commercially available from Sigma Chemical Co, St. Louis, Mo. and DifcoLaboratories Inc., Detroit, Mich.).

The present invention provides for the detection of targetmicroorganisms which express highly conserved but sterically inaccesibleantigen epitopes by combining an inventive composition of growth mediafollowed by detection with very specific antibodies using a detectionformat, such as a visual immunoprecipitation assay, enzyme linkedimmunoassay, chemiluminescence, immunoblotting, or similar technology.The present invention permits such detection by providing a growthenvironment in a modified culture medium wherein the microorganisms arepermitted to multiply to optimal levels but their surface structure isaltered, without causing substantial cell death, to expose the highlyspecific and conserved antigen epitopes found in the interior cell wallstructure.

Using the present invention the analyst can incubate the test sample ofinterest under routine laboratory conditions in the presence of theinventive growth medium which exposes the specific antigen epitopes.This invention provides a highly accurate test result while stillaffording the analyst with the convenience of standard microbiologicallaboratory conditions. A further aspect of the present invention is thatno unique or costly equipment and facilities are required.

Since continued cell viability is important to allow the pathogen ofinterest to grow to sufficient numbers for detection by the chosendetection system (e.g., visual immunoprecipitate assay, enzyme linkedimmunoassay, chemiluminescence, immunoblotting, or similarimmuno-affinity based detection technology), the present inventionutilizes methodologies which simultaneously induce altered cell wallcompositions as well as allowing for further growth of the pathogen.More specifically, the present invention is directed to a highlyspecific detection of target microorganisms by contacting samplespotentially containing these microorganisms in the presence of a growthmedium containing structure modifying organic chemicals which allow theexpression and accessibility of these highly conserved antigen epitopesto specific monoclonal or polyclonal detecting antibodies bound todetecting reagents. Detection is accomplished by means a visualimmunoprecipitate assay, enzyme linked immunoassay, chemiluminescence,immunoblotting, or similar immuno-affinity based detection technology.The present invention permits such detection by modifying the surfacestructure of the target microorganism without causing substantial celldeath in such a manner that the more highly conserved and specificantigen epitopes are made accessible to the corresponding antibodieslinked to detecting reagents.

The media composition of the present invention biochemically modifiesthe metabolism of the target microorganism so that it produces amodified cell wall which exposes the most specific and conservedepitopes. (See, e.g., Tsang et al., “Screening for Salmonella with aMurine Monoclonal Antibody M105 Detects both Felix O1 BacteriophageSensitive and Resistant Salmonella Strains,” Zbl.Bakt. 286:23-32, 1997;Tsang et al., “A Murine Monoclonal Antibody that Recognizes aGenus-Specific Epitope in the Salmonella Lipopolysaccharide Outer Core,”Zbl.Bakt. 274: 446-455, 1991; Tsang et al., “A Murine MonoclonalAntibody Specific for the Outer Core Oligosaccharide of SalmonellaLipopolysaccharide,” Infection and Immunity, 55: 211-216, 1987; Tsang etal., “Lack of the α-1,2-linked N-acetyl-D-glucosamine epitope in theouter core structures of lipopolysaccharides from the certain Oserogroups and subspecies of Salmonella enterica,” Res. Microbiol. 142:521-533, 1991). The structural modification occurs without inhibitingthe microorganisms ability to grow, therefore, the target pathogenmicroorganism continues to grow uninhibited to reach a detectable level.The combination of structural modification and the ability to furtherreplicate provides an advantage in that the pathogenic microorganismsare generally found in a sample at levels below the detection thresholdof most rapid detection systems. While any detection system may beemployed, preferred detection systems include, but are not limited to,visual immunoprecipitate assay, enzyme linked immunoassay,chemiluminescence, immunoblotting, and similar detection systems.

In one embodiment of the present invention, a test sample potentiallycontaining a pathogenic microorganism is contacted with a growth mediumcontaining an inhibitor of O-antigen polysaccharide cell surfaceexpression. Subsequently, the sample containing media is subjected to adetection methodology, which may include visual immunoprecipitate assay,enzyme linked immunoassay, chemiluminescence, immunoblotting, or similardetection systems.

In preferred embodiments, the compositions, formulae, detection devices,and the methods of detecting are specific for Listeria,Enterohemorrhagic E. coli (EHEC), Salmonella, or Campylobacter. In aparticularly preferred embodiment, the inventive growth medium,following incubation, is introduced into a detection system, such as avisual immunoprecipitate assay, an enzyme linked immunoassay,chemiluminescence, immunoblotting, or similar detection technologycontaining an antibody specific for the target microorganism therebyproducing a highly accurate result.

The present invention combines any of several widely recognized generalenrichment media such as tryptic soy broth, nutrient broth, bufferedpeptone water, lactose broth, brain heart infusion broth, or similarmedia with a number of antigen structure modifying organic chemicals,including but not limited to 2,4-dinitrophenol, carbonylcyanide-m-chlorophenyl hydrazone or similar electron uncouplers toexpose epitopes which are recognized by monoclonal or polyclonalantibodies which have high affinity for the specific epitopes. Themechanism of action of these organic chemicals is to alter the metabolicpathways of the target microorganism such that it produces a deficientcell wall allowing exposure of the interior specific epitopes. It is thecombination of the alteration of a metabolic pathway which alters thestructure of the cell wall with an antibody-detection reagent containedin a detection device such as visual immunoprecipitate assay which makesthe detection rapid and specific.

Following incubation in the inventive media under permissive conditionsthe results are detected preferably using a rapid detection method suchas, but not limited to, visual immunoprecipitate assay, enzyme linkedimmunoassay, chemiluminescence, immunoblotting, or similar detectiontechnology. Such methodologies are described in greater detail in U.S.Pat. No. 5,658,747 and PCT WO 95/30903. In a preferred embodiment of theinvention, the mixture of the composition and the test sample, followingincubation, may be exposed to a detergent solution to improve theaccessibility of the conserved antigen epitope. In a most preferredembodiment of the invention the detergent may be heated to facilitate amore rapid exposure of the epitope. In yet another embodiment, themixture, following incubation, is exposed to detergent at an elevatedtemperature, the temperature is preferably from about 40° C. to 121° C.for a specified time, preferably from two minutes to one hour.

In one embodiment, the mixture, prior to detection, compromises up toabout 0.02-2.0% by a weight of a detergent, preferably an anionicdetergent, further preferably selected the group consisting of sodiumdodecyl sulfate (SDS) and sodium deoxycholate, and the like, but alsoincluding non-ionic detergents such as NP-40, tergitol and Triton X-100,and the like.

An additional aspect of the present invention is the use of a visualimmunoprecipitate assay to detect the presence of a microorganism in atest sample. In the visual immunoprecipitate assay, the antibodies,including the “antibody-detection-reagent” initially located in thereagent zone, is typically either a polyclonal or monoclonal antibody.Further, when using a polyclonal antibody the antibody is preferablyaffinity column purified prior to its utilization the present invention.The production of such antibodies is well known in the art. (See, e.g.,Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988). Suitable affinity purified antibodiescan also be procured from commercially available sources. For example, apolyclonal antisera specific for Salmonella is available from Kirkegaardand Perry Laboratories, Gaithersburg, Md. A preferred visualimmunoprecipitate assay is that which is described by U.S. Pat. No.5,658,747. Briefly, U.S. Pat. No. 5,658,747 utilizes a lateral flowdiagnostic device which comprises a reagent zone containing anantibody-detection reagent and a detection zone located downstream ofthe reagent zone and comprising an immobile binding partner capable ofspecifically binding said complex between the target microorganism andthe antibody detection reagent.

Polyclonal antibodies can be readily generated by one of ordinary skillin the art via immunization of a variety of warm-blooded animals such ashorses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice, orrats. Briefly, the target microorganism, or an antigen specificallyassociated with the target microorganism, is utilized to immunize theanimal. The immunogenicity of the protein or peptide of interest may beincreased through the use of an adjuvant such as Freund's complete orincomplete adjuvant or by coupling to another protein such as ovalbuminor keyhole limpet hemocyanin (KLH).

Monoclonal antibodies can also be readily generated using well-knowntechniques. (See, e.g., Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow andLane (eds.), supra.) Briefly, as one example, a subject animal isimmunized as with the production of a polyclonal antibody.Alternatively, in vitro immunization techniques suitable for theproduction of monoclonal antibodies are also known in the art.Antibody-producing cells are then fused to immortal myeloma cells toprovide an immortal hybridoma cell line. Following the fusion, the cellsare placed into culture plates containing a suitable medium,traditionally HAT medium, although other suitable media are known in theart. After about seven days, the resulting fused cells or hybridomas maybe screened in order to determine the presence of antibodies whichrecognize the desired antigen. Following several clonal dilutions andreassays, hybridoma producing antibodies that bind to the protein ofinterest can be isolated.

Other techniques can also be utilized to construct monoclonal antibodiesor binding partners. (See, e.g., Huse et al., “Generation of a LargeCombinational Library of the Immunoglobulin Repertoire in Phage Lambda,”Science 246:1275-1281, 1989; Sastry et al., “Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc. Natl. Acad. Sci. USA 86:5728-5732,1989; Alting-Mees et al., “Monoclonal Antibody Expression Libraries: ARapid Alternative to Hybridomas,” Strategies in Molecular Biology 3:1-9,1990; Larrick et al., “Polymerase Chain Reaction Using Mixed Primers:Cloning of Human Monoclonal Antibody Variable Region Genes From SingleHybridoma Cells,” BioTechnology 7:934-938, 1989.)

Once a suitable antibody has been obtained, it may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Antibodies: A Laboratory Manual, Harlow and Lane, supra).

Antibodies useful in the present invention are preferably capable ofselectively detecting all of the strains of a target microorganism inthe presence of numerous antigenically related organisms. Further, theantibodies are preferably capable of such detection with a low tolerancefor non-specific binding (which leads to a false positive result) and avery low, preferably zero, failure to bind target the microorganism(which leads to a false negative result).

One aspect of the present invention provides a general enrichmentmedium, tryptic soy broth, containing 0.1-5 mM 2,4-dinitrophenol towhich a test sample is added, thereby forming a mixture, andsubsequently incubated at 37° C. for 6-8 hours. Following incubation, analiquot of the sample is exposed to 0.05-0.5% SDS at 100° C. for tenminutes. The sample may then introduced into a detection device, forexample, a visual immunoprecipitate assay device and observed for theformation of a visual line.

Preferably, the sample is a solution containing, or consistingessentially of, an unpurified field sample such as a food sample, anenvironmental sample such as water or dirt. Alternatively, the samplemay be a biological fluid such as a body fluid. In a further embodiment,the sample may be partially or substantially purified prior toadministration to the diagnostic device, such as a laboratory sample.Upon contacting the sample with a composition containing a specificantibody-detection reagent for the target microorganism that ispotentially contained within the sample, binding between theantibody-detection reagent and the target microorganism is permitted,thereby detecting the presence or absence of a particular pathogenicmicroorganism.

Another aspect of the present invention provides a method for detectinga microorganism in a test sample wherein the test sample is incubated ina general enrichment media comprising at least one structure modifyingorganic chemical for sufficient time to propagate detectable levels ofmicroorganisms. Subsequently, the presence of pathogenic microorganismsis detected by utilizing immuno-based detection methodologies, whichinclude but are not limited to, immuno-affinity, visualimmunoprecipitation, enzyme linked immunoassay, chemiluminescence,immunoblotting, and the like. Alternatively, the exposure of antigen ina sample may be enhanced by treatment with detergent prior to orcontemporaneously with detection. In a further alternative embodiment,the exposure of antigen in a sample, previously subject to propagationin the presence of the composition of the present invention, may beenhanced by heating the sample in the presence of the detergent, priorto or contemporaneously with detection.

In yet another aspect, the present invention provides methods ofdetecting a target microorganism comprising contacting a samplepotentially containing the target microorganism in the presence of othergenera not of interest but expressing cross reactive antigen epitopeswith a composition as described above under permissive incubationconditions. Following incubation the sample is exposed to an assay suchas the visual immunoprecipitate assay that permits theantibody-detection reagent to bind to the target microorganism toprovide a complex between the target microorganism and theantibody-detection reagent. The complex then migrates downstream alongthe lateral flow membrane to a detection zone containing an immobileantibody capable of binding to the complex to provide a bound complex.Next, the bound complex is detected.

The following examples are presented for the purpose of illustration,not limitation.

EXAMPLES Example 1

Nine strains of Salmonella were identified which were non-reactive in avisual immunoprecipitate assay. The strains were reported to produceexcessive levels of surface antigen. It was hypothesized that growth ofthe strains in the inventive media would eliminate or significantlyreduce the expression of surface O group antigen epitopes, therebyallowing detection by highly specific monoclonal antibodies directedagainst the core region of lypopolysaccharide contained in the visualimmnunoprecipitate assay device.

The organisms were grown in the inventive medium, followed by extractionwith 0.1% SDS at 100° C. for ten minutes. Strong reactivity wasdemonstrated. The inventive media formulation was a commerciallyavailable formulation of tryptic soy broth containing the followingingredients supplemented with 0.5 mM (0.01%) 2,4-dinitrophenol.

Trypticase Soy Broth Pancreatic Digest of Casein 17.0 g Papaic Digest ofSoybean Meal 3.0 g Sodium Chloride 5.0 g Dipotassium Phosphate 2.5 gDextrose 2.5 g Distilled Water 1000 ml

Example 2

Two strains of Salmonella, serogroups A and B, were determined to beweakly reactive in a monoclonal antibody based visual immunoprecipitateassay and an enzyme linked immunoassay in the presence of relatedcompetitive microorganisms. These strains were incubated in theinventive medium in a 1000 fold excess of competitive bacteria, andfound to be highly reactive. The inventive media formulation was acommercially available formulation of buffered peptone watersupplemented with 0.5 mM (0.01%) 2,4-dinitrophenol.

Buffered Peptone Pancreatic Digest of Gelatin 10.0 g Sodium Chloride 5.0g Disodium Phosphate 3.5 g Monopotassium Phosphate 1.5 g Distilled Water1000 ml

Example 3

A strain of E. coli 0157:H7 was found to be weakly reactive in apolyclonal antibody based assay. The strain was grown in the inventivemedium and the sensitivity was improved by 100 fold. The media was amodified tryptic soy broth with 20 mg/ml novobiocin supplemented with0.5 mM (0.01%) 2,4-dinitrophenol.

Modified Tryptic Soy Broth Bacto Tryptone 17.0 g Bacto Soytone 3.0 gSodium Chloride 5.0 g Dipotassium Phosphate 4.0 g Bile Salts No. 3 1.5 gBacto Dextrose 2.5 g

Example 4

A pathogenic strain of Campylobacter jejuni was identified which did notreact in a polyclonal/monoclonal based enzyme immunoassay. The strainwas grown in the inventive medium followed by treatment with 0.1% sodiumdexoycholate and was found to be strongly reactive. The media wasCampylobacter isolation broth supplemented with 0.5 mM (0.01 %)2,4-dinitrophenol.

Nutrient Broth No. 2 with 0.6% yeast extract Lab-Lemco Powder 10.0 gPeptone 10.0 g Sodium Chloride 5.0 g Yeast Extract 6.0 g Distilled Water1000 ml

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit an scope of the invention. Accordingly, the invention is notto be limited except as by the appended claims.

We claim:
 1. A method for detecting a microorganism in a test sample,comprising: (a) contacting a test sample with a composition comprisinggeneral enrichment media and at least one structure modifying organicchemical, thereby forming a mixture; (b) incubating the mixture for atleast 6 hours to allow for detectable levels of microorganisms todevelop, and (c) detecting the presence of specific microorganisms inthe mixture; and wherein said structure modifying organic chemical is0.1-5 mm 2,4 dinitrophenol.
 2. The method of claim 1, wherein thepreferred time sufficient to allow for detection is about 6-8 hours. 3.The method according to claim 1, wherein the microorganism is selectedfrom the group consisting of Listeria, Enterohemorrhagic E. coli,Salmonella, and Campylobacter.
 4. The method according to claim 1,further comprising contacting the mixture with a detergent, wherein saidcontact further exposes antigenic epitopes prior to detection, andwherein said detergent is present in an amount of about 0.02% to 2% byweight of detergent.
 5. The method according to claim 4, furthercomprising heating the combination of the mixture and the detergentprior to detection.
 6. The method according to either claim 4 or 5,wherein the detergent is an anionic detergent.
 7. The method accordingto claim 5, wherein the detergent is selected from the group consistingof sodium dodecyl sulfate and sodium deoxycholate.
 8. The methodaccording to claim 4 or 5, wherein the detergent is a non-ionicdetergent.
 9. The method according to claim 8, wherein the detergent isselected from the group consisting of NP-40, tergitol, and triton X-100.10. The method according to claim 5, wherein heating is performed atabout 40° C. to about 121° C. for two minutes to one hour to furtherexpose antigenic epitopes.
 11. A method for detecting the presence ofListeria, Enterohemorrhagic E. coli, Salmonella, or Campylobacter in atest sample, comprising: (a) contacting a test sample with a compositioncomprising general enrichment media and at least one structure modifyingorganic chemical, thereby forming a mixture; (b) incubating the mixturefor at least 6 hours to allow for detectable levels of microorganisms todevelop; (c) detecting the presence of specific microorganisms in themixture, wherein a positive detection result indicates the presence ofListeria, Enterohemorrhagic E. coli, Salmonella, or Campylobacter in thetest sample; and wherein said structure modifying organic chemical is0.1-5 mm 2,4-dinitrophenol.
 12. The method according to claim 11,further comprising contacting the mixture with a detergent, wherein saidcontact further exposes antigenic epitopes prior to detection, andwherein said detergent is present in an amount of about 0.02% to 2% byweight of detergent.
 13. The method according to claim 12, furthercomprising heating the combination of the mixture and the detergentprior to detection.
 14. The method according to claim 12 or 13, whereinthe detergent is an anionic detergent.
 15. The method according to claim13, wherein the detergent is selected from the group consisting ofsodium dodecyl sulfate and sodium deoxycholate.
 16. The method accordingto claim 12 or 13, wherein the detergent is a non-ionic detergent. 17.The method according to claim 16, wherein the detergent is selected fromthe group consisting of NP-40, tergitol, and triton X-100.
 18. Themethod according to claim 12, wherein heating is performed at about 40°C. to about 121 ° C. for two minutes to one hour to further exposeantigenic epitopes.
 19. The method according to claim 11, whereindetection occurs by an immunoassay.
 20. The method according to claim19, wherein the immunoassay is selected from the group consisting of avisual immunoprecipitate assay, an enzyme linked immunoassay,chemiluminescence, and immunoblotting.
 21. The method according to claim20, wherein the immunoassy is a visual immunoprecipitate assay.
 22. Themethod according to claim 20, wherein the detection utilizes acomplementary monoclonal antibody, polyclonal antibody, or an antibodyfragment, and wherein said antibody or antibody fragment is specific fora highly conserved cell wall epitope.
 23. A method for detecting amicroorganism in a test sample, comprising contacting a test samplecontaining a microorganism with an immunoaffinity based detectiondevice, wherein said test sample has been previously propagated in thepresence of a structure modifying organic chemical; and wherein saidstructure modifying organic chemical is 0.1-5 mm 2,4-dinitrophenol. 24.A method for detecting microorganism specific epitopes on a targetmicroorganism in a test sample, comprising: (a) propagating amicroorganism in a test sample in a permissive general enrichment media,wherein said media comprises a structure modifying organic chemical; (b)contacting the test sample with a microorganism specific antibody linkedto a detecting reagent, wherein reaction with the antibody indicates thepresence of the microorganism; and wherein said structure modifyingorganic chemical is 0.1-5 mm 2,4-dinitrophenol.
 25. The method of anyone of claims 1, 11, 23, and 24, wherein said test sample is selectedfrom the group consisting of a food product, water, an environmentalsample, a biological sample, a human specimen, and a veterinary sample.